Normal cells have limited proliferative potential and eventually undergo irreversible growth arrest or senescence. In contrast, cancer cells escape senescence and display cellular immortality. In human cells, escape from senescence requires inactivation of both the so-called pRB and p53 "cancer pathways". Consistent with cellular immortality being an essential phenotype of the cancer cell, it has been suggested that the pRB pathway is inactivated in virtually all human cancers. However, close analysis of the pRB pathway suggests that this is an over simplification. If so, then how do transformed cells acquire immortality? Our preliminary data suggests the existence of a novel cellular pathway that contributes to onset of cellular senescence. At the core of this pathway are two proteins previously implicated in control of cell proliferation and/or tumor suppression, namely (promyelocytic leukemia) PML and HIRA. The PML protein is fused to the retinoic acid receptor-alpha (RARalpha) in acute promyelocytic leukemias (APL) due to the t(15:17) chromosomal translocation. HIRA is the human ortholog of two repressors of histone gene transcription in yeast, Hirlp and Hir2p. We propose either that inactivation of the HIRA/PML pathway allows escape from senescence in the presence of an active pRB pathway, or that the HIRA/PML pathway is an "extension" of the pRB pathway and its inactivation essentially inactivates pRB. Accordingly, the aims of this proposal are: 1) to define the signaling relationship between the putative HIRA/PML pathway and known cancer pathways, such as the senescence-inducing pRB and p53 pathways; and 2) to test whether disruption of the HIRA/PML body association contributes to transformation of human cells. Completion of these Specific Aims should uncover an extension of a known cancer pathway or, alternatively, reveal an entirely novel cancer pathway. Either way, given its role in senescence, this pathway is likely to be a rich source of oncogenes and tumor suppressors. Undoubtedly, the first definition of this pathway and its subsequent potential for disclosure of novel diagnostic, prognostic and therapeutic cancer targets is of high impact for both our understanding of cancer biology and our ability to design therapeutic strategies.